Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD

Abstract

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

Modeling Intestinal Epithelial Response to Interferon-γ in Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids

“…While there may be conflicting data with regard to the sole addition of IFN-γ, numerous studies in adenocarcinoma lines have shown that the addition of IFN-γ with TNF-α causes mislocalization of ZO-1 and E-cadherin [ 23 , 24 , 25 ]. Although we did not examine the effect of co-administration of TNF-α and IFN-γ in this study, we did in a recently published report and found that the addition of both cytokines does cause mislocalization of ZO-1 and E-cadherin in iPSC-derived intestinal epithelium [ 26 ], and thus additionally supports our model.…”

“…IBD is a complex polygenic disorder in which over 200 disease-associated risk loci have been discovered [ 30 , 31 , 32 ] and numerous clinical subtypes of disease exist [ 33 ]. Using iPSC-derived HIOs presented here or by incorporating them onto transwells [ 26 ] or into small microengineered Chips [ 15 ], we can begin to define personalized responses to various environmental factors and cytokines and model therapeutic outcomes in a precision medicine-based manner, which ultimately may improve drug selection for the treatment of IBD.…”

M1 Macrophage exosomes MiR‐21a‐5p aggravates inflammatory bowel disease through decreasing E‐cadherin and subsequent ILC2 activation

“…Cell adhesion molecules mediate adhesion and binding between cells or between cells and the extracellular matrix. E‐cadherin is a type of cell adhesion molecule that plays an important role in maintaining integrity by mediating mutual aggregation between cells 25,26 ; however, its dysregulation has been shown to participate in the pathogenesis and remission of UC 25‐27 . In particular, the down‐regulation of E‐cadherin expression leads to the destruction of the intestinal mucosal barrier, 27 which manifests clinically as repeated bloody diarrhoea.…”

“…E-cadherin is a type of cell adhesion molecule that plays an important role in maintaining integrity by mediating mutual aggregation between cells 25,26 ; however, its dysregulation has been shown to participate in the pathogenesis and remission of UC. [25][26][27] In particular, the downregulation of E-cadherin expression leads to the destruction of the intestinal mucosal barrier, 27 which manifests clinically as repeated bloody diarrhoea. The findings of this study confirmed that Ecadherin expression was significantly down-regulated in intestinal mucosa with UC; however, it remains unclear whether abnormally polarized macrophages can regulate E-cadherin expression in the intestinal mucosa or how this process is initiated.…”

Genetic Correction of IL-10RB Deficiency Reconstitutes Anti-Inflammatory Regulation in iPSC-Derived Macrophages

“…Additionally, the barrier function of epithelial cells is impaired in certain IBD patients. These functions were so far studied in an iPSC-derived intestinal epithelial cell model in an immune cell-free environment [ 35 ]. Furthermore, Caspase 8 deficiency in three unrelated patients was associated with IBD caused by impaired control of inflammation and intestinal barrier integrity [ 36 ].…”